Advances in the study of biological molecules have been led, in part, by improvement in technologies used to characterise the molecules or their biological reactions. In particular, the study of the nucleic acids DNA and RNA has benefited from developing technologies used for sequence analysis.
Nucleic acid sequencing methods have been known in the art for many years. One of the best-known methods is the Sanger “dideoxy” method which relies upon the use of dideoxyribonucleoside triphosphates as chain terminators. The Sanger method has been adapted for use in automated sequencing with the use of chain terminators incorporating fluorescent labels.
There are also known in the art methods of nucleic acid sequencing based on successive cycles of incorporation of fluorescently labelled nucleic acid analogues. In such “sequencing by synthesis” or “cycle sequencing” methods the identity of the added base is determined after each nucleotide addition by detecting the fluorescent label.
In particular, U.S. Pat. No. 5,302,509 describes a method for sequencing a polynucleotide template which involves performing multiple extension reactions using a DNA polymerase to successively incorporate labelled polynucleotides complementary to a template strand. In such a “sequencing by synthesis” reaction a new polynucleotide strand based-paired to the template strand is built up in the 5′ to 3′ direction by successive incorporation of individual nucleotides complementary to the template strand. The substrate nucleoside triphosphates used in the sequencing reaction are labelled at the 3′ position with different 3′ labels, permitting determination of the identity of the incorporated nucleotide as successive nucleotides are added.
The guanine base of DNA is known to act as a quencher of some fluorophores, meaning that a fluorophore attached to G is harder to detect than the equivalent fluorophore attached to C, A or T (Torimura et al., Analytical Sciences, 17: 155-160 (2001); Kutata et al., Nucleic Acids Res., 29(6) e34 (2001)). In the context of a sequencing reaction based on detection of fluorescent labelled nucleotides, this in turn means that the fluorescent signal detected from labelled guanine nucleotides incorporated during the sequencing reaction will be of lower intensity than that detected from labelled nucleotides bearing the same fluorophore attached to adenine, thymine or cytosine containing nucleotides. Thus, in certain circumstances the presence of a “G” nucleotide may be harder to call with certainty than the presence of A, T or C under the same reaction and detection conditions.
Accordingly, in the context of nucleic acid sequencing reactions it would be desirable to be able to increase the intensity of the fluorescent signal from fluorescently labelled G nucleotides so that the intensity of the signal compares more favourably with that which can be obtained from fluorescently labelled A, T or C nucleotides under the same reaction and detection conditions.